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Abstract

In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4⁺CD49b⁺LAG-3⁺ T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25⁺ Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10⁺Foxp3⁻CD4⁺ T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

Keywords

Tr1 cell melanoma granzyme B perforin mechanism

Introduction

Malignant melanoma is a highly aggressive skin cancer, with a 1-year survival rate of 25% in metastatic patients and limited treatment options. Many lines of evidence have indicated that macrophages in malignant melanoma contribute to tumor progression. Tumor-associated macrophages (TAMs) represent the most abundant immune cell type in melanoma lesions.^1,2 Increased TAM activity is associated with worse prognosis and poorer response to treatments.^3–5 Multiple macrophage-produced growth factors, cytokines, and chemokines are implicated in the initiation, growth, and metastasis stages of melanoma and in the suppression of the immune system to assist melanoma cell survival. For example, TAM-derived interferon gamma (IFN-γ) promotes the aberrant growth and migration of ultraviolet (UV)-irradiated melanocytes and increases the growth and survival of melanoma cells.⁶ The production of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1α by TAMs increases melanoma angiogenesis.^7,8 Furthermore, inhibition of CD8⁺ T cell–mediated cytotoxicity by macrophages establishes a suppressive microenvironment for the infiltrating immune cells.^9–12 These results suggest that future therapeutic options may need to include the inhibition of TAM activities.

The type 1 regulatory T (Tr1) cells represent a CD4⁺ T-cell subset with well-known roles in suppressing adaptive immune responses and establishing peripheral tolerance.^13,14 Unlike conventional CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells, Tr1 cells do not constitutively express CD25 or Foxp3 and are most commonly identified by their IL-10^hiTGF-β⁺IL-2^lowIL-4⁻IFN-γ^low cytokine expression profile.^14,15 Tr1 cells can suppress activated immune cells through high IL-10 and transforming growth factor beta (TGF-β) secretion.¹⁴ In addition, Tr1 cells prevent immune activation by eliminating antigen-presenting cells of myeloid origin through the secretion of granzyme B and perforin,^15–17 thus preventing further activation of antigen-specific T cells. The role of Tr1 cells in melanoma has not been studied in detail.

We are particularly interested in the interaction between Tr1 cells and TAMs in metastatic melanoma patients. Although Tr1 cells are primarily associated with immunosuppression, which is generally considered tumor-promoting, their capacity to eliminate macrophages, one of myeloid immune cells with antigen-presenting function, made us wonder whether Tr1 cells could eliminate the tumor-promoting TAMs in melanomas and result in better melanoma control. We therefore examined this hypothesis in the following study.

Materials and methods

Patients and tumor samples

Patients with metastatic melanoma (n = 14) were diagnosed and recruited at The First People’s Hospital of Jining City. Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis, isolated by standard Ficoll-Paque PLUS (GE Healthcare) centrifugation, and stored in 90% fetal bovine serum (FBS)–10% dimethyl sulfoxide (DMSO) at −80°C. Resected tumor from the same patients was dissociated with mechanical dissection followed by enzymatic digestion.¹⁸ Tumor mononuclear cells (TMCs) were then isolated by Ficoll-Paque PLUS centrifugation, washed in complete RPMI 1640 media, and used fresh. All sample collection and experimentation procedures were approved by the Ethics Board of The First People’s Hospital of Jining City. Written consent form was obtained from each participant.

T-cell subset isolation

To fractionate CD4⁺ T cells into various CD49b versus LAG-3 subsets, CD4⁺ T cells from PBMCs were first isolated using the EasySep Human CD4⁺ T Cell Enrichment Kit (Stemcell) and then stained with anti-CD49b (BioLegend) and anti-LAG-3 (eBioscience) mAbs for sorting inside a BD FACSAria cytometer. Naive CD4⁺ T cells were isolated from PBMCs using the EasySep Human Naive CD4⁺ T Cell Enrichment Kit (Stemcell).

Flow cytometry

Cells were first washed in cold staining buffer (phosphate-buffered saline (PBS) supplemented with 2% FBS) and then incubated in dark with surface antibodies for 30 min on ice. Excess antibodies were removed by washing in staining buffer twice. Intracellular/nuclear staining was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Commercially available anti-human CD3, CD4, CD49b, LAG-3, IL-10, Foxp3, granzyme B, and perforin mAbs (from BioLegend or eBioscience) were used. Dead cells were excluded using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific).

Luminex assay

The human IL-10, granzyme B and perforin secretion was detected by combining corresponding magnetic beads from HCYTOMAG-60K and HCD8MAG-15K kits (EMD Millipore) following manufacturer’s instructions. During incubation, cells were cultured at the bottom compartment, while the beads were placed at the top compartment, separated by a 1.0 µm pore-sized insert in a Transwell plate. Beads were harvested after 3 days for IL-10 and 24 h for granzyme B and perforin.

Real-time polymerase chain reaction

Messenger RNA (mRNA) was extracted from CD4⁺ T-cell subsets using the RNeasy Mini Kit (Qiagen), and complementary DNA (cDNA) was synthesized using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The level of cDNA was detected using the SYBR Green PCR Master Mix, the AmpliTaq Gold DNA Polymerase, and the Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). Values were standardized according to the 18S ribosomal RNA (rRNA) levels using the 2^−ddCt formula. The primers used were as follows: IL-10—forward 5′-GTGGAGCAGGTGAAGAATGC-3′ and reverse 5′-ATAGAGTCGCCACCCTGATG-3′ and Foxp3—forward 5′-TTCGAAGAGCCAGAGGACTT-3′ and reverse 5′-ATGGCACTCAGCTTCTCCTT-3′.

Macrophage differentiation from peripheral blood monocytes

Modified melanoma-conditioned media was made according to a published protocol with minor modifications.¹⁹ Briefly, human malignant melanoma cell line A375 (Sigma-Aldrich) was seeded in a T-75 flask and incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM l-glutamine and 10% FBS for 3 days. The supernatant was concentrated 40 times and was added to RPMI 1640 complete media (supplemented with 10% FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU penicillin, 100 µg/mL streptomycin, and 100 µM β-mercaptoethanol) at 1:80 ratio to make the modified melanoma-conditioned media. For macrophage differentiation, monocytes were first obtained from PBMCs using the EasySep Human Monocyte Isolation Kit (Stemcell) and then added to a six-well plate in 4 mL modified melanoma-conditioned media at 2 × 10⁶ cells per well; 50% media was replaced every 3 days. After 7 days in a 37°C CO₂ incubator, the macrophages were harvested after being incubated for 15 min at 37°C in Cell Dissociation-PBS Solution Non-enzymatic (Sigma).

Proliferation assay

An amount of 50,000 A375 cells per well was placed at the bottom chamber of a 0.4 µm Transwell-24 plate (Corning), either alone or with 0.2×, 1×, or 5× as many macrophages (i.e. 1 × 10⁴, 5 × 10⁴, or 2.5 × 10⁵ cells per well, respectively) at the top chamber. After 3 days, the top chamber was removed, and the proliferation of A375 cells was assayed using the WST-1 Cell Proliferation Reagent (Abcam) and measured in a microplate reader at optical density (OD) 450 nm (Bio-Rad).

Cytotoxicity assay

Sorted Tr1 cells (CD49b⁺LAG-3⁺ in CD4⁺ T cells) or naive CD4⁺CD45RA⁺ T cells were activated by 10 µg/mL anti-CD3 mAb, 2 µg/mL anti-CD28 mAb, and 100 U/mL recombinant IL-2 (BioLegend) for 3 days, after which the cells were washed and incubated with ⁵¹Cr-labeled tumor-promoting macrophages at various concentrations, with Z-Ala-Ala-Asp chloromethyl ketone (Z-AAD-CMK; EMD Millipore) in some experiments, and the ⁵¹Cr release after 4 h was measured as previously described.²⁰ For the examination of granzyme B and perforin from T cells, Tr1 or naive T cells were added to unlabeled macrophages and were subsequently isolated by Human CD4⁺ T Cell Enrichment Kit (Stemcell). For perforin inhibition, Tr1 cells were incubated with concanamycin A (CMA; Santa Cruz Biotechnology) for 2 h before addition to macrophages.

Statistical analyses

Data were represented as mean ± standard error of mean (SEM) when applicable. The statistical differences between multiple groups were tested by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post-tests. The p value less than 0.05 was considered significant. All tests were performed in Prism software (GraphPad).

Results

Tr1 cells were enriched in the CD49b⁺LAG-3⁺CD4⁺ T-cell subset

To enable the isolation of human Tr1 cells, we first tried to identify the surface marker–associated IL-10⁺Foxp3⁻ Tr1 cells, but not Foxp3⁺ conventional Treg cells. It was previously demonstrated that human Tr1 cell clones presented elevated CD49b and LAG-3 gene expression, which was maintained in Tr1 cells developed from in vitro anergized cell culture.²¹ In addition, it was demonstrated by flow cytometry that most CD25⁻CD44^hi Tr1 cells aggregated in the CD49b⁺LAG-3⁺ fraction (>50%), while most conventional Treg cells were CD49b⁻LAG-3⁻ (56.4%).²² Therefore, we examined whether CD49b and LAG-3 could be used as an isolation marker for Tr1 cells in melanoma patients. CD4⁺ T cells were sorted into CD49b⁻LAG-3⁻, CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺ fractions on flow cytometry (Figure 1(a)), and then, each subset was activated using anti-CD3/CD28 mAbs and IL-2 and incubated in a Transwell system with anti-IL-10 beads at the top and cells at the bottom. IL-10 expression in each subset was examined on Day 3 by Luminex. Similar to previous findings, the CD49b⁺LAG-3⁺CD4⁺ T-cell subset secreted more IL-10 than other CD4⁺ T-cell subsets (Figure 1(b)). This preferential expression of IL-10 in CD49b⁺LAG-3⁺CD4⁺ T cells was confirmed by real-time polymerase chain reaction (PCR) analysis, in which we found that IL-10 mRNA was most abundantly expressed by CD49b⁺LAG-3⁺CD4⁺ T cells (Figure 1(c)). The Foxp3 mRNA level was also examined. We found that the Foxp3 mRNA expression was concentrated in the CD49b⁻LAG-3⁻ and CD49b⁺LAG-3⁻ subsets, but not in the CD49b⁻LAG-3⁺ or CD49b⁺LAG-3⁺ cell subsets (Figure 1(c)). These results together illustrated that in the peripheral blood of melanoma patients, CD49b⁺LAG-3⁺ could be used to isolate CD4⁺ T cells that were Foxp3⁻ and IL-10⁺, which are hallmarks of Tr1 cells.

Figure 1.

CD49⁺LAG-3⁺CD4⁺ T cells represent the Tr1 cells in melanoma patients. Cytokine detection was performed in duplicates and mRNA measurement was performed in triplicates (N = 14). (a) Representative gating of peripheral blood CD4⁺ T cells by CD49 and LAG-3, which was used for the sorting of CD49b⁻LAG-3⁻, CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺ subsets. (b) The IL-10 secretion in each of the CD49b⁻LAG-3⁻, CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺CD4⁺ T-cell subsets from PBMCs, examined by Luminex, after 3-day activation with anti-CD3/CD28 mAbs and IL-2 (***p < 0.001). (c) The IL-10 and Foxp3 mRNA copy numbers in each of the CD49b⁻LAG-3⁻, CD49b⁺LAG-3⁻, CD49b⁻LAG-3⁺, and CD49b⁺LAG-3⁺CD4⁺ T-cell subsets from PBMCs after 3-day activation with anti-CD3/CD28 mAbs and IL-2 (*p < 0.05; **p < 0.01).

Primary Tr1 cells eliminated tumor-promoting macrophages

TAMs are abundant in melanoma tumors, tend to prime regulatory responses, and are associated with worse prognosis in multiple studies. Tr1 cells could eliminate myeloid antigen-presenting cells through granzyme B and perforin, thus preventing antigen presentation and enhancing peripheral tolerance.^16,17,23 We wondered whether Tr1 cells had a role in eliminating regulatory macrophages. To examine this, we first made tumor-promoting macrophages that functionally and phenotypically resembled the TAMs in melanoma, by culturing peripheral blood monocytes in modified melanoma-conditioned media as previously described.¹⁹ To demonstrate that these macrophages possessed tumor-promoting capacity, we cocultured macrophages and melanoma cell line A375 in separate compartments of a Transwell plate with a 0.4 µm membrane to physically separate direct contact while allowing the transmission of soluble factors. The melanoma cells with higher numbers of macrophages presented significantly better growth, thus demonstrating the tumor-promoting capacity of these macrophages (Figure 2).

Figure 2.

Macrophages derived in modified melanoma-conditioned media possessed tumor-promoting effects. A375 human melanoma cells were placed at the bottom chamber of a Transwell plate, while 0 (A375 only), 0.2×, 1×, or 5× macrophages were placed at the top for 3 days. The macrophages were derived from peripheral blood monocytes in modified melanoma-conditioned media. The amount of A375 cell growth was measured by the standard WST-1 assay and expressed as fold relative to the A375 only condition (N = 14). Each condition was performed in triplicates (*p < 0.05; ***p < 0.001).

Next, we examined the possibility that Tr1 cells could eliminate tumor-promoting macrophages. Autologous Naive CD4⁺ T cells (isolated from CD4⁺ T cells using CD45RA⁺ marker) or Tr1 cells (isolated from CD4⁺ T cells using CD49b⁺LAG-3⁺ marker) were first activated by anti-CD3/CD28 mAbs and IL-2 and then cocultured with tumor-promoting macrophages that were developed in modified melanoma-conditioned media at various effector-to-target ratios. Naive CD4⁺ T cells presented very limited cytotoxicity, while the Tr1 cells effectively eliminated macrophages (Figure 3(a)). The secretion of granzyme B and perforin by Tr1 cells and naive T cells was then examined by Luminex assay. Tr1 cells secreted significantly more granzyme B and perforin than naive T cells (Figure 3(b)). Interestingly, Tr1 cells that were cultured alone had less perforin secretion than Tr1 cells that were cultured with macrophages, suggesting that Tr1 cells by themselves had moderate cytolytic activity but required the presence of target cells for maximal activation.

Figure 3.

Tr1 cells killed tumor-promoting macrophages through granzyme B- and perforin-mediated mechanisms. Measurement of cytotoxicity and granzyme B and perforin secretion were performed in duplicates. All cells were harvested from PBMC (N = 14). (a) The specific lysis of tumor-promoting macrophages mediated by activated Tr1 cells versus activated naive CD4⁺ T cells at various effector (T cell) to target (macrophage) ratios (***p < 0.001). (b) The granzyme B and perforin secretion by activated Tr1 and naive CD4⁺ T cells, incubated alone or after cytotoxicity assay with macrophages, examined by Luminex (*p < 0.05; **p < 0.01; ***p < 0.001). (c) The specific lysis of tumor-promoting macrophages by activated Tr1 cells in the presence of various concentrations of granzyme B inhibitor Z-AAD-CMK or after pretreatment of Tr1 cells with perforin inhibitor CMA (***p < 0.001).

The mechanism of Tr1 cell–mediated macrophage killing was then investigated. Z-AAD-CMK, a granzyme B inhibitor, was added to the Tr1 cell and macrophage coculture at various concentrations. We found that Z-AAD-CMK at concentrations greater than 10 µM resulted in significantly decreased specific lysis of macrophages by Tr1 cells (Figure 3(c)). In other experiments, activated Tr1 cells were first pre-incubated with 100 nM of the perforin inhibitor CMA, washed, and then added to autologous tumor-promoting macrophages in cytotoxicity assays. Pre-incubation of Tr1 cells with CMA also abrogated Tr1 cell–mediated cytotoxicity (Figure 3(c)). Together, these results indicated that Tr1 cells mediated cytotoxicity through granzyme B and perforin.

Tr1 cells inhibited melanoma cell growth through reducing the number of tumor-promoting macrophages

Next, we examined the effect of Tr1 cells on melanoma cells. In the previously established macrophage–melanoma cell coculture system (Figure 2), we added various amounts of activated Tr1 cells to the top chamber, where tumor-promoting macrophages were present. The number of Tr1 cells was expressed as a ratio relative to the number of macrophages, that is, if no macrophage was added, the Tr1 cells were also absent. We found that in the absence of Tr1 cells, more tumor-promoting macrophages resulted in higher A375 growth. But when high numbers of Tr1 cells (3:1) were added to the coculture, the tumor-promoting effects of macrophages were abrogated, evidenced by the lack of increase in A375 growth (Figure 4(a)). To examine whether Tr1 cells directly induced melanoma cell death, we cultured various concentrations of activated Tr1 cells at the top chamber in the absence of macrophages, while the A375 cells were placed at the bottom. No obvious difference in A375 growth was observed without or with Tr1 cells (Figure 4(b)). Together, these results suggested that Tr1 cells inhibited melanoma cell growth promoted by macrophages, but were unable to inhibit melanoma cell growth by themselves.

Figure 4.

Tr1 cells, but not conventional Treg cells, inhibited melanoma cell growth mediated by tumor-promoting macrophages. T cells and tumor-promoting macrophages were sourced from PBMCs. Each condition was performed in duplicates (N = 14). (a) The level of A375 cell growth at the bottom chamber of a Transwell plate in the absence or presence of various concentrations of macrophages and Tr1 cells in the top chamber (*p < 0.05; **p < 0.01; ***p < 0.001). (b) The level of A375 cell growth in the absence or presence of various concentrations of Tr1 cells (NS: not significant). (c) The level of A375 cell growth in the absence or presence of macrophages and CD25⁺ Treg cells (NS: not significant).

Conventional Treg cells did not share the same tumor-inhibiting effects of Tr1 cells

Foxp3 mRNA could be detected in the CD49b⁺LAG-3⁺CD4⁺ T-cell subset, albeit at reduced levels (Figure 1(c)), suggesting that conventional CD25⁺Foxp3⁺ Treg cells were present at low frequencies in the CD49b⁺LAG-3⁺ fraction. Since conventional Treg cells were thought to have tolerance-inducing roles similar to Tr1 cells,²⁴ we investigated whether the CD25⁺Foxp3⁺ Treg cells could mediate similar antitumor effects as Tr1 cells. CD25⁺ T cells (Treg) were sorted from CD4⁺ T cells and were added to top chamber of the previously described macrophage–melanoma cell coculture system. No inhibition of macrophage-mediated tumor-promoting effect was observed with the addition of Treg cells (Figure 4(c)), thus demonstrating a remarkable functional difference between conventional Treg cells and Tr1 cells.

IL-10⁺Foxp3⁻CD4⁺ T cells were present in resected tumors, with low granzyme B and perforin expression

Our investigations so far had focused on the peripheral blood Tr1 cells. To examine the Tr1 cells directly acting on the tumors, we investigated the presence and function of tumor-infiltrating Tr1 cells. Since the amount of TMCs was limited, we were unable to sort CD4⁺ tumor-infiltrating T cells according to their CD49b and LAG-3 expression while still having enough cells in each subset for Luminex assay. Instead, the TMCs were examined by flow cytometry for the identification of IL-10⁺Foxp3⁻CD4⁺ T cells and the granzyme B and perforin expression (Figure 5). We found that IL-10⁺Foxp3⁻CD4⁺ T cells could be found in TMCs, representing between 2.6% and 20.1% of total CD4⁺ T cells, which was significantly higher than in PBMCs (Figure 5(b)). However, these IL-10⁺Foxp3⁻CD4⁺ T cells from TMCs contained significantly lower frequencies of granzyme B⁺ and perforin⁺ cells than their counterparts from PBMCs (Figure 5(d)).

Figure 5.

IL-10⁺Foxp3⁻CD4⁺ T cells were present in resected tumors but had low granzyme B and perforin expression (N = 14). (a) Representative gating of IL-10⁺Foxp3⁻CD4⁺ T cells in TMCs in one metastatic melanoma sample. (b) The frequency of IL-10⁺Foxp3⁻CD4⁺ T cells as a percentage of total tumor-infiltrating CD4⁺ T cells in TMCs and PBMCs (***p < 0.001). (c) The expression of granzyme B and perforin by IL-10⁺Foxp3⁻CD4⁺ T cells in one representative metastatic melanoma sample. (d) The frequency of granzyme B-single positive (Granzyme B+ Only), perforin-single positive (Perforin+ Only), and granzyme B-/perforin-double positive (Granzyme B+ Perforin+) cells as a percentage in tumor-infiltrating IL-10⁺Foxp3⁻CD4⁺ T cells in TMCs and PBMCs (**p < 0.01; ***p < 0.001).

Discussion

Our main finding in this study was that Tr1 cells in metastatic melanoma patients were capable of suppressing the macrophage-mediated tumor cell growth through the elimination of macrophages, which suggested a novel antitumor role of Tr1 cells in melanoma. To demonstrate this, we used CD49b⁺LAG-3⁺ phenotype as the surface marker to isolate Tr1 cells from peripheral blood CD4⁺ T cells. These Tr1 cells were then incubated with tumor-promoting macrophages. We showed that compared to naive CD4⁺ T cells, the Tr1 cells presented significantly stronger potency in eliminating macrophages. At 10:1 effector:target ratio, greater than 30% target macrophages were killed by Tr1 cells, while at 3:1 effector:target ratio, approximately 20% macrophages were killed by Tr1 cells. Tr1 cells secreted abundant levels of granzyme B and perforin when incubated with tumor-promoting macrophages. Inhibition of granzyme B and perforin in Tr1 cells significantly decreased the specific lysis of macrophages. When incubated with tumor-promoting macrophages at the top chamber of a Transwell, the melanoma cell line A375 at the bottom chamber presented significantly better growth, but when Tr1 cells were placed at the top chamber together with the macrophages, the increase in A375 cell growth was partially impaired when the Tr1:macrophage ratio was 1:1 and was completely abrogated when the Tr1:macrophage ratio was 3:1. The final result was surprising because previously, we showed that Tr1 cells at 3:1 effector:target ratio were only responsible for the killing of approximately 20% of macrophages. This seemingly amplified efficacy of Tr1 cells in abrogating macrophage-mediated tumor growth was possibly due to the longer incubation time of 3 days in the A375 cell proliferation assay, as compared to the 4-h incubation time in the macrophage lysis assay, allowing more extensive elimination of macrophages.

We also observed another apparent discrepancy between different experiments. Tr1 cells incubated in the absence of macrophages were capable of secreting granzyme B and perforin, but were incapable of directly eliminating A375 cells. Several factors possibly contributed to this observation. First, to prevent mismatches in major histocompatibility complex (MHC) molecules, the primary Tr1 cells and A375 cells were separated across a membrane, which allowed the transmission of granzyme B and perforin but disallowed direct cell-to-cell contact. It was previously demonstrated that Tr1 cell–mediated killing of myeloid cells required human leukocyte antigen (HLA) class I molecules recognition and lymphocyte function–associated antigen 1 (LFA-1) adhesion on the target cells,¹⁶ which were not possible in the experimental Transwell system. Second, we showed that Tr1 cells incubated with macrophages presented higher perforin secretion than Tr1 cells incubated alone, suggesting that some macrophage-mediated effects might be necessary for the full activation of Tr1 cell cytotoxicity. And third, it was observed that Tr1 cell–mediated killing was specific to cells of myeloid origin, but not cells of lymphocytic or erythroblastic origins.¹⁶ The specific underlying mechanisms for this phenomenon are unknown, but it was shown that the expression of CD54, CD58, CD155, and CD112 on myeloid target cells was necessary.¹⁶ These cell surface molecules are possibly required for a more stable adhesion between effectors and targets and better activation of the effectors.^25–27

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms